1. Field of the Invention
The present invention relates to a prothrombin time (PT) reagent using purified, reconstituted natural or recombinant human tissue factor (rTF). More particularly, the invention relates to the reconstitution of tissue factor (TF) into phospholipid vesicles or micelles to produce a tissue factor-based PT reagent. Such a reagent allows specific monitoring of oral anticoagulant therapy and deficiencies in the extrinsic pathway of coagulation.
2. Description of Related Art and Introduction to the Invention
In general, liposomes are a general category of vesicle which comprise one or more lipid bilayers surrounding an aqueous space. Within this category are unilamellar vesicles composed of a single membrane or lipid bilayer, and multilamellar vesicles composed of many concentric membranes (or lipid bilayers). Liposomes are commonly prepared from phospholipids. Szoka, F. and Papahadjopoulos, D., Ann. Rev. Biophys. Bioeng., 9:467-508 (1980). Micelles are different from liposomes. Micelles form when molecules possessing both hydrophobic and hydrophilic properties, such as detergents, are put into aqueous media. The hydrophobic portions of the molecules aggregate to avoid the aqueous media. In their simplest state, micelles may be spherical; however, they may form aggregates (bilayers) of various shapes and sizes. Micelles differ from liposomes in having a hydrophobic interior rather than an aqueous interior. For example, the hydrophilic heads of the detergent molecules comprising the micelle face outward into the water while the hydrophobic tails join company with other like hydrophobic structures. Davis, B. D. and Dulbecco, R. "Sterilization and Disinfection." Microbiology, 3rd Edition, p 1270, Harper & Row (Davis, B. D., et al., 1980); Mahler, H. R. and Cordes, E. H., Biological Chemistry, Second Edition, Harper & Row Publishers, pp. 712-714 (1971).
Liposomes have been used as a drug delivery system. This approach takes advantage of the fact that liposomes have a relatively impervious lipid bilayer which may enclose an interior aqueous space and thereby provide a method to completely encapsulate various drugs within this interior space. Szoka, supra, at p. 468. An important aspect of a delivery system of this type would be that the active ingredient drug was unavailable to the aqueous medium outside the liposome until it reached its target. Janoff et al., U.S. Pat. Ser. No. 4,880,635 (1989).
It has been observed that the tissues of vertebrates, when added to citrated plasma and recalcified, will profoundly accelerate clotting time. This tissue constituent which has been observed to activate the coagulation protease cascades is commonly referred to as thromboplastin or tissue factor (TF).
In 1935, the use of thromboplastin (procoagulant tissue factor) was first described in a one stage PT test (Quick, J. Biol. Chem., 109:73-74, 1935). This test employed thromboplastin derived from mammalian tissue and a standard curve prepared with dilutions of pooled normal human plasma. The modern version of this test is easy to perform and can be automated.
The prothrombin time (PT) test is the most commonly performed assay in the coagulation laboratory. Variants of this test have a number of uses (White, et al., Hemostasis and Thrombosis, Basic Principles and Clinical Practice, Coleman, et al., eds., J. B. Lippencott Co., Philadelphia, pp. 1048-1060, 1987). One use is to assess deficiencies in the extrinsic pathway of coagulation (factors VII, X, V, and prothrombin). A second use is to monitor patients undergoing long term oral anticoagulant therapy for disorders such as recurrent venous thrombosis and cancer (Hirsh, J., Seminars in Thrombosis and Hemostasis, 12:1-11, 1986). A third use is to evaluate liver dysfunction.
The therapeutic range of anticoagulant therapy is based on the avoidance of bleeding and thrombolic complications. When monitoring oral anticoagulant therapy, as well as for a variety of other conditions by the PT test, an elongation of prothrombin time by a factor of 2 is most desirable for long term therapy (O'Reilly, Hemostasis and Thrombosis, Basic Principles and Clinical Practice, Coleman, et al., eds., J. B. Lippencott Co., Philadelphia, pp. 1367-1372, 1987). This elongation factor is defined as the prothrombin ratio (PR) and is calculated by dividing the PT of a patient plasma by the PT of a pool of plasmas from normal individuals. A higher PR indicates a more sensitive PT reagent. The benefits of a more sensitive reagent for monitoring anticoagulation therapy is the use of lower doses of anticoagulant drug. These lower doses still provide adequate protection against thromboembolic disease while minimizing bleeding complications.
Several reagents for determining PTs are commercially available. These include Thromborel S (Curtis Matheson Scientific, Inc., Yorba Linda, Calif.) and Simplastin (Organon Teknika Corp., Charlotte, N.C.). These reagents yield very different PTs for the same patient plasma, with Thromborel S exhibiting a longer time than Simplastin. Lower doses of anticoagulant drug are therefore required to maintain extended PT times (high PR) when the PTs are monitored using Thromborel S instead of Simplastin. A need exists for an even more sensitive tissue factor based PT reagent to monitor anticoagulant therapy and other conditions. The present invention provides just such a sensitive reagent with its highly desirable PR.